1. Field of the Invention
The present invention relates to methods and vectors for making specific mutations in genes. More specifically, the invention relates to the use of a vector system useful in modifying the eukaryotic genome, particularly of embryonic stem cells, at precise and predefined loci by the means of homologous recombination.
2. Description of the Related Art
Many different technologies have been described that lead to chromosomal alterations and thereby to a modification of the structure and/or expression of genes. One technique for targeted mutagenesis is based on homologous recombination. The general methodologies of targeting mutations into the genome of cells, and the process of generating mouse lines from genetically altered embryonic stem (ES) cells with specific genetic lesions are well known (Bradley, 1991,Cur. Opin. Biotech. 2: 823-829).
A synthetic recombination vector which contains the genetic information of the targeted chromosomal locus recombines with the genomic DNA after introduction into a cell. The vector usually contains a positive selection cassette which is flanked by the genetic information of the target locus to enrich for cells where the vector successfully recombines with the chromosomal DNA against the pool of non-recombinant cells. Stable integration leads to a long term resistance against certain pharmacological toxins. Examples are the resistance against G418, i.e., Geniticin, or Hygromycin by the action of the neomycin or hygromycin resistance genes, respectively. The position of the positive selection cassette in the chromosomal vector DNA can further lead to a mutation of the gene as in classical knockout experiments, i.e., inactivation of gene function. Furthermore, inactivation or modification of regulatory elements of the target gene as well as of domains of the transcribed/translated gene product could have positive, negative or modulatory effects on future target gene function.
Homologous recombination, that is carried out by the target DNA flanking the positive selection cassette, has to be selected against the background of unwanted non-homologous recombination that is thought to occur over the vector ends. A negative selection cassette positioned at the terminus of the vector will frequently be integrated by the non-homologous recombination events. Stable expression of the negative selection marker leads to cytotoxicity of otherwise non-cytotoxic agents. An example is the activated cytotoxicity of Gancyclovir by the action of the Herpes Simplex virus thymidine kinase gene product (HSV-TK). The likelihood of obtaining a homologous recombination event increases with the size of the chromosomal vector DNA and is further dependent on the isogenicity between the genomic DNA of the vector and the target cell.
The cloning of large chromosomal fragments (5,000-15,000 base pairs) of the target gene, the subcloning of this DNA into a bacterial plasmid vector, the mapping of the gene structure, the integration of the positive selection cassette into the vector and finally, the flanking of one or both homologous vector arms by a negative selection marker is a technically demanding task and generally requires long construction times (3-6 months). The construction of the recombination vector itself is, therefore, most often the time limiting step in targeted mutagenesis experiments.
The prior art is deficient in the lack of effective means of constructing vectors for homologous recombination directed mutagenesis. The present invention fulfills this longstanding need and desire in the art.